The Metabolic Biochemistry of The
c.l
THE METABOLIC BIOCHEMISTRY OF THE
WANDERING SHREW (SOREX VAGRANS)
by
BRIAN EMMETT
B. Sc., Dalhousie University, Halifax, 1.97^
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in the
DEPARTMENT OF-ZOOLOGY
We accept this thesis as conforming to the
required standard
i THE UNIVERSITY OF BRITISH COLUMBIA i October, 1980 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study.
I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of
The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5
Date Ot4~ H- 11*30 i
ABSTRACT
The comparative examination of the metabolism of the shrew, Sorex vagrans, at the ultrastructural, mitochondrial and enzymatic level has re• vealed a number of consistent factors which correlate with the high basal metabolic rate of these mammals. The metabolic picture which emerges is of an organism highly dependent on aerobic metabolism, utilizing lipid or
fatty acids as a primary energy source. This appears particularly true
for skeletal muscle which, in larger mammals, may be fueled primarily by glycogen and depend more upon anaerobic metabolism at high work loads.
Ultrastructural studies reveal that the diaphragm and gastrocnemius muscle are composed of small diameter fibers, associated with an abundance
of peripheral and interfibrillar mitochondria. These typical red fibers
are considered to operate aerobically, either in a fast or slow twitch
fashion. Levels of oxidative (citrate synthase, fumarase 8-OH butyrylCoA
dehydrogenase) enzymes are elevated with respect to the rat in heart,
liver and gastrocnemius muscle while glycolytic enzymes (glycogen phos- phorylase, PK, LDH) are depressed in all tissues but heart which has PK and
LDH activities comparable to rat heart.
No large differences were observed in the absolute rates or substrate
preferences of shrew and rat cardiac mitochondria but enzymatic profiles
of these organelles ..show increased activities of citrate synthase and
B-OH butyrylCoA dehydrogenase in shrew mitochondria. This may indicate in•
trinsic differences between shrew and rat cardiac mitochondria, the physio•
logical consequences of which remain to be elucidated.
Glycogen is relatively scarce in shrew tissues and, although present
in liver, does not form typical mammalian liver a-rosettes. The low levels of phosphorylase in the tissues assayed indicate that the shrew has a re• duced ability to metabolize this substrate. In contrast lipid stores appear abundant in all tissues examined.
In the gastrocnemius muscle of a series of seven mammals ranging in size from 0.004 to 400 kg., the activities of oxidative enzymes scale with respect to body mass with similar exponents as the scaling of maximal oxygen consumption (VQ^max/M^). In contrast levels of glycolytic enzymes increase as body size increases, indicating that burst anaerobic work is functionally more important in larger mammals. iii
TABLE OF CONTENTS
page
Abstract i
List of Tables v
List of Figures vi
Acknowledgements vii
Chapter 1: Introduction 1
Chapter 2: Materials and Methods 7
Experimental animals 7
Electron microscopy 7
Enzyme extraction and assay procedures 9
Preparation of mitochondria 11
Chapter 3: Ultrastructural Studies 13
Introduction 13
Results and Discussion ..... 13
Heart 13
Gastrocnemius muscle 16
Diaphragm 23
Liver 27
Chapter 4: Mitochondrial Respiration of Shrew Cardiac Mitochondria. 32
Introduction 32
Results and Discussion 36
Chapter 5: Enzyme Profiles in Various Shrew Tissues 42
Introduction 42
Results and Discussion 43
Heart 43 iv
Page
Liver 46
Skeletal muscle 47
Scaling of gastrocnemius muscle enzyme acti• vities 48
Chapter 6: Discussion , 57
Literature Cited 67 V
LIST OF TABLES
Table Page
I Respiration rates of mitochondria from heart tissue of
the shrew and rat 37
II Enzyme activities in mitochondria prepared from shrew
and rat hearts 38
III Enzyme activities in the heart, skeletal muscle and
liver of the shrew and rat 44
IV Enzyme activities in the gastrocnemius muscle of
animals of varying size 49
V Statistical analysis of linear regressions of enzyme
activity versus body mass 55 vi
LIST OF FIGURES
Figure Page
1 Electron micrograph of shrew cardiac tissue 14
2 Electron micrograph of shrew heart muscle showing the
association of lipid with interfibriliar mitochondria . . 15
3 Electron micrograph of gastrocnemius muscle.fibers of
the shrew 17
4 Longitudinal section of shrew gastrocnemius muscle ... 19
5 Frequency distribution of gastrocnemius fiber diameter
from the shrew 21
6 Electron micrograph of shrew diaphragm 24
7 Frequency distribution of diaphragm muscle fiber diameter
from the shrew . • 25
8 Representative electron micrograph of shrew hepatocytes . 28
9 Activity of citrate synthase in the gastrocnemius muscle
of several mammals as a function of body size 51
10 Activity of lactate dehydrogenase in the gastrocnemius
muscle of several mammals as a function of body size . . 52 vii
ACKNOWLEDGEMENTS
This work was supported by an NSERC - (Canada) Operating Grant to
Peter Hochachka. To Peter and all the members of the.lab I am indebted
for countless discussions of shrews and scaling.as well as other topics
both biological and biochemical. Mary Taitt and Charlie Krebs provided many of the shrews-and-voles used-in this study. The technical assistance
and.expertise of Laslo Veto in the preparation of-the electron micro•
graphs is greatly .appreciated. I am also indebted, to Tom Shields for his
dogged pursuit of the .run -on-sentence in editing this -manuscript. Robin,
Risa and Tom Mommsen .provided'just the right-blend .of quiet support and
verbal chastisement to encourage me to .complete the task. Finally
Vancouver Island and Conifer Cottage provided an environment which eased
the pains.of writing, making it an enjoyable and .rewarding experience. 1
CHAPTER I
Introduction 1A
The shrews (order Insectivora, family Soricidae) are the smallest members of the mammalian class. Most species range in weight from 4 to
10 grams, and the smallest living mammal, the Etruscan shrew (Suncus etruscus), weighs only 2 grams. In contrast, the largest living mammal, g the blue whale, can weight up to 100,000 kilograms, or 2 x 10 times as mu:ch as the Etruscan shrew.
Across this mammalian size range the basal metabolic rate is related to body size by the well known expression:
= 0.676 M^-75 (1) where VQ is the basal oxygen consumption in liters 0^ h * and M^ is the body mass in kilograms. Dividing this equation by the body mass (M^) allows one to express the relationship between metabolic rate and body mass on a mass specific basis:
0,25 VQ2/Mb = 0.676 Mb" (2)
VQ /Mb is a mass specific metabolic rate (expressed in units of liters
O2 h ^ kg and to avoid using the term interchangably with the true metabolic rate (VQ^), Kleiber (1975) has suggested that Vp^/M^ be called metabolic turnover rate to emphasize its true physiological meaning as a measure of the turnover rate of chemical energy in an animal's body.
Observed rates of basal oxygen consumption for shrews are often higher than predicted by equation.2 (Vogel, 1976). The metabolic turn• over rate of a five gram shrew has been measured as 7.40 liters 0^ kg ^ h
(Hawkins et al., 1960), while an elephant's mass specific basal oxygen -1 -1
consumption is approximately 100 times less, 0.07 liters 02 kg h
(Brody, 1945). The difference in 02 consumption between a shrew and the common laboratory rat is approximately ten fold. Thus, on average, one 2
gram of shrew tissue receives and-metabolizes 10 times the amount of sub• strate and oxygen as a rat and 100 times the amount of an elephant. Such a comparison is however not strictly correct as not all types of tissue are a constant proportion of body weight. For example, the percentage of body mass which is bone increases as body mass increases to overcome the added stresses and strains large size imposes on the skeleton. Certain organs, such as liver and kidney, decrease in proportional size as body mass increases. Nevertheless, these structural differences cannot alone account for the observed differences in metabolic rate, and thus certain tissues and organs in shrews must metabolize substrate at much higher rates than their counterparts in larger mammals.
This high metabolic turnover rate has resulted in a number of morpholo• gical and physiological adaptions in small mammals in general and shrews in particular. These adaptations serve to enhance substrate and oxygen as• similation, delivery and uptake to and by the body tissues. Pernetta (1976), working from literature values, reports that species of the genus Sorex ingest 0.5 to 3 times their body weight daily. The same author measured gut retention time for ingested material in Sorex araneus. Although re• taining material in its gut for only 20 minutes to three hours, this shrew has an assimilation efficiency of up to 90%.
Oxygen uptake and delivery is facilitated in small mammals in a variety of ways. Weibel (1979) has studied the alveolar surface of the Etruscan shrew using the scanning electron microscope. The alveoli of shrews are very much smaller than larger mammals, affording a much larger surface area per unit volume (eight times that of human lung) for gas exchange. Morrison et_ al_. (1959) report that lung weight in Sorex cinerius is 1.40 times 3
higher than that predicted by the standard allometric equation,
0 99
WL = 0.0124 Mb " (see Stahl, 1967). These authors also report the re• spiration rate in this species to be very close to the heart rate (800 min ^) whereas most mammals have a resting respiratory rate one third the basal heart rate. This result is however most likely stress-induced as
Weibel (1979) measured the respiratory rate of the Etruscan shrew at
300 min * with a corresponding cardiac rate of 1050 min Nevertheless these values are astonishingly high.
No comprehensive study of capillarization of shrew tissue has been at• tempted, however it is likely that capillary density is inversely related to body size, at least in skeletal muscle. Schmidt-Nielsen and Pennycuik
(1961) report that capillary density in both red and white fibres of gastro•
cnemius is higher in smaller mammals although no systematic relationship
is evident. The oxygen capacity of blood in Suncus etruscus is 24.2 mL
O2/IOO mL blood (Bartels et al., 1979), which is close to the upper limit
recorded for mammals. This increased oxygen capacity in shrews is probably
due to a high hematocrit of 50-55%, high hemoglobin concentration, (Wolk,
1974; Bartels et^ al., 1979) and the small size (5 y) of shrew erythrocytes.
(Wolk, 1974) which affords an increased surface area for oxygen diffusion
to and from hemoglobin. In addition, the Bohr effect in shrews is even
more pronounced than other small mammals (Bartels et^ al_. , 1969) . Thus
the unloading of oxygen to rapidly metabolizing tissues, which produce
large amounts of acidic end products (lactate, Cf^), is enhanced.
It is evident that considerable adaption has occurred to enable the
cell of a rapidly metabolizing tissue in a small mammal to receive oxygen
and substrate at enhanced rates. Are there any corresponding adaptations
in cellular structure and biochemistry to aid the cell in metabolizing 4
these substrates more rapidly? Indeed is this type of adaptation necessary in order for the shrew to process substrate at such high rates? In other words is metabolic turnover rate limited strictly by delivery and uptake of substrate and oxygen or is there also a consequental change in the bio• chemical system which processes the substrate?
A number of studies have been conducted on the ultrastructure of skeletal muscle and its variation with body size. These will be reviewed in the introduction to the section on shrew ultrastructure (Chapter III).
No systematic comparative ultrastructural studies exist for other mammalian tissues. Several authors report an increase in certain enzyme activities in various tissues as body size decreases. This relationship is seen for carbonic anhydrase in mammalian erythrocytes (Larimer and Schmidt-Nielsen,
1960); cytochrome oxidase in liver (Kunkel and Campbell, 1952) and heart
(Simon and Robin, 1971); succinate dehydrogenase and malate dehydrogenase in kidney, heart, liver and brain (Fried and Tipton, 1953). Except for the work with carbonic anhydrase these studies examined only a limited number of mammalian species (3-5) and none included a member of the shrew family. The limited biochemical work which has ibeen conducted on shrews is restricted to an examination of seasonal variation in certain bio• chemical parameters, and contains little comparative information (Hyvarinen,
1968; Hyvarinen, 1979; Hyvarinen and Pasanen, 1973).
The purpose of the present study is to examine several aspects of the biochemistry of the wandering shrew, Sorex yagrans to determine if any adaptation related to the high metabolic turnover rate of this organism has occurred at the biochemical level.
This species occurs throughout British Columbia and western Alberta, northward to the Arctic coast and southward into the United States. The 5
local sub-species Sorex vagrans vagrans: is smaller than most races, averaging 9-11 cms (40% of which is tail) and weighs 4-6 grams, although both males and females may increase to 8 grams during the breeding season.
There is considerable seasonal variation in color, ranging from sooty black in winter to pale brown in summer. This shrew inhabits damp areas such as the edges of marsh, along ditches and under logs. They appear not to be commonly found in heavily wooded areas but may retreat to such a habitat in summer, when the ditches and marshes peripheral to these areas dry up. Food consists primarily of small insects and earthworms.
Shrews are known as annual mammals, rarely living much beyond one year.
Breeding generally.begins in February and offspring (average litter size is six) are born in April and May. The adults die shortly after reproducing and the offspring reach sexual maturity the following winter.
Sorex vagrans is a member of the subfamily Soricinae. This group of shrews is characterized by a basal metabolic rate even higher than pre• dicted by the allometric relationship for BMR . (Vogel, 1976). Except for the desert shrew, Notiosorex crawfordi, which enter daily bouts of shallow torpor (Lindstedt, 1980), this group of shrews does not exhibit any annual hibernation or daily torpor. Sorex vagrans is active both night and day, with peaks of activity at dusk and dawn.
The approach taken in the present study is strictly comparative and whenever feasible, concurrent determinations were carried out on the labor• atory rat, which displays a metabolic turnover rate approximately 10 fold lower than this shrew species. In order to gain a broad overview of meta• bolic organization in the shrew, this study was divided into three primary components: 6
(1) Electron microscopic examination of shrew heart, liver, diaphragm and gastrocnemius.
(2) Determination of mitochondrial respiration rates and mitochondrial substrate preferences of mitochondria isolated from shrew and rat hearts.
(3) Determination of glycolytic, Krebs cycle, and fatty acid oxidation enzyme activities in a number of shrew and rat tissues.
The specific rationale for chosing each of these approaches will be dealt with in the introductions to subsequent chapters of this thesis, which treat each of these primary components in turn. 7
CHAPTER II
Materials and Methods 7A
EXPERIMENTAL ANIMALS
Shrews (Sorex vagrans), deer mice (Peromyscus maniculatus), and voles
(Microtus townsendii) were live trapped using Longworth traps set on the
University of British Columbia Endowment Lands or at Iona Island, in the
Fraser River delta. Samples of cow muscle were kindly provided by Richmond
Meat Packers, Richmond, British Columbia. Rats, guinea pigs, and rabbits were obtained from the Animal Care Centre, U.B.C.
Animals were held in captivity for up to one week prior to use.
Shrews were maintained in a healthy state on a diet of beef heart (approxi• mately one body weight equivalent of beef heart per animal per day).
ELECTRON MICROSCOPY
For electron microscopy samples of liver, heart, diaphragm, and gastro• cnemius muscle were quickly excised from freshly killed shrews, immersed and finely chopped in 100 mM sodium/potassium phosphate buffer, pH 7.4, containing 2.5% glutaraldehyde and 2.0% paraformaldyhyde. Following the two hour fixation period, the tissues were washed three times with 100 mM phosphate buffer, pH 7.4 (adjusted with sucrose to an osmolarity of 520 milliosmols). The samples were than postfixed for one hour with 1.5% 0s0^ prepared in the phosphate buffer, washed three times in distilled water, and stained with 2.5% uranylacetate. The tissue pieces were dehydrated in a. graded ethanol series and treated with a series of propylene oxide to facilitate the penetration of Epon. The.tissues were embedded in Epon 812 according to Luft (1961). All operations, except the final curing of Epon, were carried out at room temperature. 8
Ultrathin sections were cut using glass knives fitted to a Porter-Blum
MT-2 ultramicrotome, and stained with lead citrate (Reynolds, 1963). The
sections were examined with a Zeiss EM 1G.
Mitochondrial abundance was estimated by determining the area of
electron micrographs occupied by mitochondria using a zero-compensating 2
planimeter. The total tissue area analyzed exceeded 1,500 y for each
determination. Mitochondrial abundance is expressed as the percentage of
this total tissue area occupied by mitochondria. Admittedly this esti•
mation of mitochondrial abundance is only semiquantitative as it assumes
mitochondria to be randomly distributed throughout the tissue. As shrew
gastrocnemius muscle contained such a profusion of peripheral mitochondria,
distributed in a non-random fashion, mitochondrial abundance was not measured
in this tissue. Quantitative stereological methods have been developed to
estimate morphometric parameters from microscopic studies (Weibel et_ al, 1969). Using these methods, mitochondrial volume density (volume of mito- 3
chondria/cc of tissue) is determined using a lattice of test points on the
electron micrograph and counting the faction of points enclosed within the
mitochondria. Volume density is calculated by assuming a standard ellip•
soidal shape for the mitochondria. Using these methods, a large tissue
area can be more rapidly examined than direct measurement of mitochondrial
area, thus reducing the bias introduced by the non-random distribution of
mitochondria.
For light microscopy thick sections (1 ym) were cut from the muscle
tissue blocks using a Porter Blum MT-1 ultramicrotome. The sections were
stained with 5% toluidine blue. For each muscle, sections were cut from a minimum of four blocks taken from different areas of the muscle. Fiber
diameters were determined by examining these sections with a Ziess light 9 microscope equipped with a calibrated ocular micrometer. In the case of l fibers cut in tangental section the minimum fiber diameter was measured.
A minimum of 100 fibers was measured in each tissue.
ENZYME EXTRACTION AND ASSAY PROCEDURES
For shrew and rat enzyme profile studies, tissues were rapidly removed from animals killed by cervical dislocation, rinsed in ice cold homogeni- zation buffer (50 mM imidizole> pH 7.4, containing 50 mM KC1, 7. mM MgCl,
5 mM EDTA). Tissues were blotted dry on filter paper, weighed and homo• genized in 10 volumes of homogenization buffer (using a small ground glass homogenizer). Samples were then briefly sonicated (two times 15 second bursts) and centrifuged at 3,000g for 20 minutes on a Sorvall RC2-B re• frigerated centrifuge. The supernatants were then used directly for the enzyme assays. Tissues used for the comparative study of enzyme activities of gastrocnemius muscle were treated in a similar fashion except that homo• genization was carried out on a Polytron PCU-2-110 tissue processer, with• out further sonication.
Enzyme activities were determined using a Unicam SP1800 recording spectrophotometer equipped with a thermostated cell holder maintained at
37°C by a Lauda constant temperature bath. The reaction rate was deter• mined by the increase or decrease in the absorbance of NADH or NADPH at
340 nm. Citrate synthase (Srere, 1969), carnitine acetyltransferase and carnitine palmitoyltransferase (Foster and Bailey, 1972) were monitored at
412 nm using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB). Fumarase acti• vity was determined directly by measuring the absorbance of fumarate at
240 nm (Bergmeyer, 1974). Enzyme activity is expressed as units per gram 10 fresh weight, where one unit equals 1 uM of substrate converted to product per minute.
The conditions for the enzyme assays were taken from a variety of standard procedures in the literature and, for the sake of completeness, are briefly summarized here. Most of these conditions are identical to those used by Hochachka et^ al. (1978). Unless indicated the assays were done in 50 mM imidazole buffer, pH 7.4, containing 50 mM KCL and 7 mM. MgCl.
GLYCOGEN PHOSPHORYLASE, EC 2.4.1.1: 50 mM sodium phosphate buffer, pH 7.0, 0.4 mM NADP, 4 yM glucose-1,6-diphosphate, 10 mM MgCl, 2 mg/mL gly• cogen (omitted for control), excess phosphoglucomutase and excess glucose-6- phosphate dehydrogenase. 1.6 mM AMP was added to the assay to determine
TOTAL PHOSPHORYLASE activity.
HEXOKINASE, EC 2.7.1.1 (HK): 1 mM NADP, 1 mM ATP, 1 mM glucose (omitted for control) and excess glucose-6-phosphate dehydrogenase.
PYRUVATE KINASE, EC 2.7.1.40 (PK): 0.2 mM NADH, 5 mM ADP, 5 mM phos- phoenolpyruvate (omitted for control) and excess lactic dehydrogenase.
LACTIC DEHYDROGENASE, EC 1.1.1.27 (LDH): 0.2 mM NADH, 10 mM pyruvate,
(omitted for control).
CITRATE SYNTHASE, EC 4.1.3.7: 50 mM Tris buffer, pH 8.1, 0.1 mM DTNB,
0.5 mM oxaloacetate, 0.3 mM acetylCoA (omitted for control).
FUMARASE, EC 4.2.1.2: 50 mM sodium phosphate buffer, pH 7.0, 40 mM malate (omitted for control).
MALATE DEHYDROGENASE, EC 1.1.1.37 (MDH): 0.2 mM NADH, 0.5 mM oxalo• acetate (omitted for control).
B-OH BUTYRYLCoA DEHYDROGENASE, EC 1.1.1.35: 0.2 mM NADH, 0.1 mM aceto- acetyl-CoA (omitted for control).
i 11
GLUTAMATE - OXALOACETATE TRANSAMINASE, EC 2.6.1.1 (GOT): 0.2 mM NADH,
40 mM aspartate, 7 mM a-ketoglutarate (omitted for control) and excess malic dehydrogenase.
GLUTAMATE - PYRUVATE TRANSAMINASE, EC 2.6.1.2 (GPT): 0.2 mM NADH, 200 mM alanine, 7 mM a-ketoglutarate (omitted for control) and excess lactic dehydrogenase.
GLUTAMATE DEHYDROGENASE, EC 1.4.1.3 (GDH): 0.2 mM NADH, 50 mM NH Cl,
1 mM ADP, 7 mM a-ketoglutarate (omitted for control).
CARNITINE PALMITOYLTRANSFERASE (CPT): 50 mM Tris buffer, pH 8.1, 0.1 mM
DTNB, 30 yM palmitoylCoA and 2 mM carnitine (omitted for control).
CARNITINE ACETYLTRANSFERASE, EC 2.3.1.7 (CAT): 50 mM Tris, pH 8.1,
0.1 mM DTNB, 0.2 mM acetylCoA, 2 mM carnitine (omitted* for control).
PREPARATION OF MITOCHONDRIA
Mitochondria were isolated from shrew and rat hearts by differential centrifugation, according to the method of Chappell and Hansforth (1972).
All procedures took place at 0°C and acid washed glassware was used through• out the preparation.
Hearts from three to six shrews or one to two rats were pooled and rinsed thoroughly in ice cold preparative medium (5 mM Tris, pH 7.4 at 23°C, containing 0.21 M mannitol, 0.07 M sucrose, and 1 mM EGTA). The tissues were then finely chopped in fresh medium, containing 0.2 mg Nagarase per gram of heart tissue. After rinsing with buffer, the tissues were partially homogenized in a loose-fitting Potter-Elvehjem homogenizer with fresh medium and Nagarase, and left to incubate five minutes at 0°C. The tissues were then rehomogenized until a uniform homogenate was obtained (5-10 passes of 12
the Potter-Elvehjem pestle). This homogenate was then centrifuged for 10 minutes at 10,000g to remove the proteolytic enzymes (Nagarase) and the re• sulting pellet resuspended in fresh preparation medium and spun 10 minutes at 400g to remove cellular debris. The supernatant was decanted and respun
10 minutes at 10,000g to obtain a mitochondrial pellet. The pellet was then washed twice by resuspension and repetition of the final centrifugation step. The resulting mitochondrial pellet was suspended to a final protein concentration of approximately 2 mg/mL in fresh preparative buffer con• taining bovine serum albumin (1 mg/mL).
Rates of oxygen consumption were measured using a Gilson oxygen electrode in a 2 mL glass chamber, which was maintained at 25°C by a Lauda circulating constant temperature bath. The electrode was connected to a Gilson oxygraph unit. In all cases the incubation medium contained 20 mM sodium phosphate buffer, 10 mM Tris, 100 mM KC1, 5 mM MgCl and 2.7 mg/mL bovine serum albumin
(BSA), adjusted to a pH of 7.4.
Mitochondrial protein was measured by a modified Biuret method (Gornall et_ al., 1949), in which 0.8% deoxycholate was included to ensure that mito• chondrial membranes were completely dissolved. Bovine serum albumin was used as a standard. 13
CHAPTER III
Ultrastructural Studies 13A
INTRODUCTION
Ultrastructural studies can provide important evidence regarding physio•
logical and biochemical adaptations at the cellular level. The fiber type, mitochondrial content and endogeneous storage substrate of a muscle cell
can vary according to the extent to which the cell is dependent on aerobic
or anaerobic metabolism as an energy source. Such changes are readily vis•
ible at the electron microscopic level.
An ultrastructural study of heart, liver, diaphragm and gastrocnemius muscle of the shrew, was undertaken for three reasons:
(1) To examine mitochondrial abundance, structural com• plexity, and arrangement within the tissue.
(2) To determine the type and amount of endogenous sub• strate in the tissues examined.
(3) To observe skeletal muscle ultrastructure, including the distribution of fiber type and fiber diameter.
RESULTS AND DISCUSSION
Heart
Figure 1 is a typical section of shrew cardiac tissue. Lipid droplets are very.abundant and generally juxtaposed to the mitochondria (Figure 2).
Such an arrangement of lipid stores and mitochondria is characteristic of cardiac muscle, however lipid is not nearly as abundant in the heart of
larger mammals (McNutt and Fawcett, 1974). Cardiac muscle usually contains a significant amount of glycogen, distributed as B-particles throughout the
sarcoplasm, often in the region of the I band. Glycogen, although present,
is nowhere abundant in shrew cardiac tissue and it appears that this organism 14
FIGURE 1
Electron micrograph (x 6,800) of shrew cardiac tissue. A muscle fiber is shown in both tangental (lower right) and transverse (center left) sections. Note the arrangement of mitochondria (M) between the myofibrils, abundance of lipid (L), and the scarcity of glycogen. Micrographs of a higher magnification reveal some glycogen granules but they are nowhere abundant in shrew cardiac tissue. Peripheral mitochondria are less pro• fuse in heart than in gastrocnemius muscle (Figure 3). RBC, red blood cell. 14A 15
FIGURE 2
Electron micrograph (x 43, 750) of shrew heart muscle, showing the asso• ciation of lipid (L) with interfibrillar mitochondria (M). Note the com• plex and dense arrangement of the mitochondrial cristae. 15A 16
relies almost exclusively on lipid as an endogenous fuel for cardiac meta• bolism.
Approximately 30% of the tissue section area is occupied by mitochondria.
In most mammals mitochondria occupy one quarter to one third of the myo•
cardial cells, and in the smallest mammal, the 2-3 gram Etruscan shrew,
55% of cardiac cell area is made up of mitochondria (Weibel, 1979). Thus
Sorex vagrans, although in the upper range for mammalian species, does not display an extraordinary abundance of cardiac mitochondria. McNutt and
Fawcett (1974) note that the inner mitochondrial membrane is folded into a
complex fenestrated cristae in rapidly beating hearts. Shrew heart mito•
chondria fit this pattern and it is plausible that the consequent increased
surface area of the inner membrane affords a larger area for membrane-
associated enzymes, such as those of oxidative phosphorylation and possibly the Krebs cycle.
Gastrocnemius Muscle
Figure 3 is a section through a shrew gastrocnemius muscle fiber. Peri• pheral or subsarcolemmal mitochondria, typical of red muscle fibers, are abundant. Gustafsson et_ al_. (1965) observed an increase in peripheral mito• chondria of rat gastrocnemius fibers following L-thyroxine induced increases
in basal metabolic rate. Following endurance training, the crossectional area occupied by subsarcolemmal mitochondria of rat soleus muscle increased
53% (Muller, 1976). This author hypothesizes that the interfibrillar and peripheral mitochondria serve two different functions; the former supplying the ATP required for muscle contraction, while the latter supply the energy
for the active transport of metabolites through the sarcolemma and also for the synthetic activities of the muscle fibers. As it is predominantly the 17
FIGURE 3
Electron micrograph (x 6,800) of gastrocnemius muscle fibers of the shrew.
Note the profusion of mitochondria on the periphery of the muscle fibers
(P.M.) and the presence of lipid droplets associated both with the peri• pheral mitochondria and the interfibrillar mitochondria. Glycogen,- although nowhere abundant, appears to be most closely associated with the peripheral mitochondria (see Figure 4). N, nucleus; RBC, red blood cell. 17A 18
peripheral mitochondria which increase with endurance training, Muller con•
cludes that energy supply for active transport of metabolites is the limiting
factor for soleus muscle performance under sustained aerobic conditions. In
support of this hypothesis Gollnick (1978) argues that the transport capacity
of the circulatory system generally exceeds the capacity of skeletal muscle to extract and metabolize substrate. He thus concludes that the rate-
limiting step in the uptake of fuel by skeletal muscle lies in the active transport systems of the sarcolemma.
The proliferation of peripheral mitochondria in shrew gastrocnemius is
consistent with such a theory. With decreasing animal size and increasing metabolic turnover rate, an increase in the abundance of peripheral mito•
chondria in skeletal muscle would be expected.
Figure 4 is a longitudinal section of the myofibrillar region of shrew
gastrocnemius. The arrangement of interfibrillar mitochondria adjacent to the I bands is typical of mammalian and avian red muscle fiber (Weibel,
1979). It is, however, unusual to see such a uniform and symmetrical dis• tribution of interfibrillar mitochondria in the I band region. Such an arrangement is similarly pronounced in the flight muscles of hummingbirds, where mitochondria form a distinct, almost solid band between adjacent myo•
fibrils (Drummond, 1971). This type of mitochondrial arrangement is usually assumed to be functionally important. Weibel (1979) speculates that the mitochondria are so arranged to facilitate the diffusion of 0^ and ATP
through the comparatively loose structure of actin filaments in the I-band region.
Lipid seems to be the predominant endogenous storage form of energy.
Lipid droplets, however, are not nearly as abundant in shrew gastrocnemius as cardiac muscle. The lipid droplets are associated with both peripheral 19
FIGURE 4
Longitudinal section (x 17,000) of shrew gastrocnemius muscle. Note the symmetrical arrangement of mitochondria (M) between the I bands of each myofibril. Endogenous substrate is not as plentiful as cardiac tissue
(Figure 1), however some lipid (L) is present adjacent to the mito• chondria and glycogen is scarce in the interfibrillar area. A; A-band;
Z, Z-line; M1, M-line. 19A and interfibrillar mitochondria. Electron micrographs of a higher magni• fication than those included here reveal evidence of glycogen in both the peripheral and interfibrillar regions. It is however scarce, and appears to be more closely associated with the peripheral region (Figure 3). As endo• genous substrate stores are low in shrew gastrocnemius compared to the heart it is probable that this muscle relies more heavily on blood-born substrates
(glucose or free fatty acids) during periods of normal aerobic function.
The gastrocnemius is regarded as a muscle of mixed fiber type (Henneman and Olson, 1965; Romanul, 1964). In the rat the axial portions of both the medial and lateral gastrocnemius are visibly redder than the superficial portions (Romanul, 1964). It is therefore expected that the fiber diameter distribution of this type of muscle should display a wide and possibly bi- phasic pattern. Unfortunately most analyses of gastrocnemius fiber com• position report only mean diameter or percent composition of fiber types.
Hegartyand Hooper (1971) report mouse gastrocnemius fiber distribution as monophasic but wide ranged (20-120 ym), while Goldspink (1962) found mouse gastrocnemius to have a biphasic fiber distribution.
Figure 5 depicts the frequency distribution of muscle fibers in the gastrocnemius muscle of shrews, measured from muscle sections cut for light microscopy from Epon-embedded tissues. The range of fiber diameters (10-55 um) is less than that reported for mice and corresponds to the lower half of the distribution range. The mean of all fibers measured is 28.7 +_ standard error of 0.5 ym. Joubert (1956) reports a mean fiber diameter of 73 ym for cow, 50 ym for sheep, 91 ym for pig and 76 ym for rabbit gastrocnemius.
Shafiq et al_. (1969) report a median fiber diameter of 40 ym for mouse gastrocnemius while Hegarty and Hooper (1971) give a mean value of 21
FIGURE 5
Frequency distribution of gastrocnemius fiber diameter from the shrew.
Data pooled from two animals. Mean fiber diameter +_ S.E. is indicated.
N = 239. FREQUENCY ( Percent ) ro 01 o o O
on
«1
si
ro
Tl Ol
CD m 70 > s PT H m 3)
IT 3 2 > n ro a>
M- Ol o
3 si
g-H 23
60 um for this muscle in the same organism. The latter authors used pre• parations from unfixed, unembedded, rigor muscles which are less susceptible to shrinkage. For such scant data, it is difficult to determine any
systematic variation in gastrocnemius muscle fiber diameter with decreasing body size; however the mean gastrocnemius fiber diameter in the shrew is smaller than any value which could be found for this muscle in the liter• ature. The muscle fibers are relatively uniform in size and in the smaller
(red muscle) range of mammalian muscle fiber size.
Diaphragm
The arrangement of interfibrillar mitochondria and lipid droplets in shrew diaphragm (Figure 6) resembles gastrocnemius muscle except that the mitochondria are ,more irregularly spaced and larger, and lipid droplets are more abundant. Glycogen granules are again rare. Although not particularly evident in this electron micrograph, peripheral mitochondria are very much in evidence, often associated with cell nuclei.
Mitochondria were calculated to occupy 31% of the sectional area in these electron micrographs. Mitochondria make up 15% of rat diaphragm
(Weibel, 1979). Thus, as opposed to cardiac tissue, an increase in mito• chondrial content of diaphragm muscle with decreasing body size is observed.
Diaphragm, like gastrocnemius muscle, is composed of both red and white fibers and thus the relative proportions of fiber types can change in re• sponse to selective pressure originating from varying physiological demands.
This adaptive strategy is ruled out in the case of cardiac muscle since it is not differentiated into metabolically specialized cell types.
Figure 7 shows the frequency distribution of fiber diameter in shrew 24
FIGURE 6
Electron micrograph (x 13,500) of shrew diaphragm. In comparison with shrew gastrocnemius skeletal muscle (Figure 4), it appears that the interfibrillar mitochondria are more irregularly spaced, larger and more abundant in diaphragm muscle. In addition, the amount of endogenous lipid is greater in diaphragm. 24A 25
FIGURE 7
Frequency distribution of diaphragm muscle fiber diameter from the shrew.
Data pooled from two animals. Mean fiber diameter +_ S.E. is indicated.
N = 105. FREQUENCY .( Percent ) ro o O o _i_ _l_
O
ro. -TI Ol CD 70 m
O " > m H m 70
m > o z ro
Ol
•c 3 Ol o
Ol Ol
9Z 27 diaphragm. The diameters range from 15-45 ym with a mean of 27.0 ym (+_ standard error of 0.7 ym). In a comparative study of mammalian diaphragm,
Gauthier and Padykula (1966) report the mean fiber diameter of the diaphragm of the shrew, Blarina brevicauda, to be 18 ym. As in gastrocnemius muscle, the range of diaphragm muscle fiber diameters is narrow in Sorex vagrans and corresponds to the diameter range of typical red fibers. In their study
Gauthier and Padykula conclude that the diaphragm of small mammals is com• posed of uniformly small (average diameter 22 ym) fibers rich in mitochondria and lipid droplets. The diaphragm of large mammals (80 kg and up) is com• posed of uniformly large (52 ym) fibers in which mitochondria and lipid are scarce. Mammals of intermediate size (65 gm to 80 kg) have diaphragms made up of a heterogeneous mix of these two fiber types. The diaphragm of the shrew, Sorex vagrans, fits this general descriptive pattern of fiber com• position in mammalian diaphragm.
Liver
Shrew liver is composed of hepatocytes rich in mitochondria and trigly• ceride droplets. In some cells, lipid droplets appear to occupy one third to one half of the cell volume (Figure 8). As in other mammalian species, the inner membrane of shrew liver mitochondria is less highly folded than muscle mitochondria. Glycogen is readily observable although, unlike most mammalian livers, it is present as 8-particles (20-50 nm) rather than a-particles (rosette-like aggregations of glycogen, 40-200 nm in size). Gly• cogen rosettes are presumably efficient ways of storing large quantities of glycogen (Hochachka and Hulbert, 1978) and the absence of a-particles in shrew liver is indicative of a decreased dependence on glycogen metabolism in this animal. 28
FIGURE 8
Representative electron micrograph (x 17000) of shrew hepatocytes. A profusion of lipid (L) is evident as well as glycogen (G) scattered throughout the cytoplasm. Micrographs of a higher magnification confirm that this glycogen is of the monoparticulate 6 form. N, nucleus; M, mitochondria; RER, rough endoplasmic reticulum. 28A 29
In summary, the examination of shrew tissues at the electron micro• scopic level has revealed a number of clues as to the metabolic strategy of this group of animals. In all tissues examined lipid appears to be the predominate endogenous storage form of energy. Glycogen is rare in heart and skeletal muscle tissues and in liver is present only as B-particles and not packaged into the more highly structured a-rosettes normally found in mammalian liver.
It is however difficult to obtain quantitative comparative information on lipid stores and it is known that lipid in the liver of shrews may in• crease during captivity (Sicart £t al., 1978). These same authors did not find any significant differences in the lipid content of Suncus etruscus liver and livers of mice, rats or rabbits. In addition, overall body fat content in shrews is no higher than values measured for rodents (Myrcha,
1969) and the only significant fat reserves are found in the brown adipose tissue. Malzahn (1974) reports that the levels of brown adipose tissue in the shrew, Sorex araneus, are from four to ten times higher than the vole,
Clethrinomys glaneolus. However, brown adipose tissue is primary metabolized in situ, to provide a non-shivering source of heat (Smith and Roberts, 1964), and it is unlikely that this tissue would supply large amounts of substrate to other body tissues. It is likely that a large proportion of the lipids metabolized by the shrew is provided as blood-born free fatty acids.
Mitochondria are also abundant in all tissues examined, especially in skeletal muscle (diaphragm and gastrocnemius). Peripheral mitochondria are abundant in these skeletal muscles, consistent with the concept that the energy supplied by these mitochondria is used for the active transport of metabolites to the muscle fibers and that this energy supply is rate limiting for sustained aerobic work (Muller, 1976). If such a hypothesis is correct, 30
shrew skeletal muscle appears to be adapted to function particularly well
in a sustained oxidative fashion.
In the shrew both diaphragm and gastrocnemius, muscle are composed of
homogeneous populations of small diameter fibers, rich in lipid and mito•
chondria. Although only these two skeletal tissues wer^: examined ultra-
structurally, it appears that shrew skeletal muscle is composed primarily
of red-type fibers geared toward oxidative function. In larger animals,
the fiber composition of gastrocnemius is often reported as a heterogeneous mix of fiber types (Romanul, 1964; Henneman and Olsen, 1965). Few studies have been conducted to determine whether any systematic variation in muscle fiber composition occurs with decreasing body size. The work of
Gauthier and Padykula (1966) indicates that differences do occur in the
fiber composition of diaphragm, which in intermediate sized mammals is composed of a heterogenous mixture of fiber types. A comparable study by
Davies and Gunn (1972), utilizing histochemical techniques, indicates an
increase in the abundance of fast-twitch fibers of reduced anaerobic capa•
city in the diaphragm of small animals. The same authors observed similar
results in a comparative study of semitendinosus muscle (Davies and Gunn,
1971). From these limited studies, it appears that smaller animals tend
to show an increase in the relative amount of red (oxidative)fibers in
muscles which are usually regarded as being composed of a mixture of fiber
types. Both the gastrocnemius and diaphragm of shrews appear to fit this model aptly.
The distinction between red and white muscle is, in some sense, quite
arbitrary. Most investigators recognize at least three types of muscle
fibers in mammals (Henneman and Olson, 1965; Gauthier, 1970; Weibel, 1979)
although as many as eight have been described (Romanul, 1964). Most mam- 31
malian muscles are composed of varying ratios of these three muscle types
(alternatively called red, intermediate and white fibers or slow twitch- oxidative, fast twitch-oxidative and fast twitch-glycolytic). Some muscles, including gastrocnemius, may vary in their fiber composition in different portions of the muscle (Romanul, 1964). A muscle can thus be composed of any combination of these three fiber types resulting in a potential con• tinuum of muscle structure and function from fast glycolytic to slow sus• tained oxidative. If diaphragm and gastrocnemius can be taken as exemplary of skeletal muscle, then the shrew undoubtedly lies on the red (oxidative) end of the spectrum compared to mammals of larger size.
It thus appears that the shrew has evolved a metabolic strategy based on the oxidative metabolism of fatty acids. The complete oxidation of fat to carbon, dioxide and water yields, on a weight basis, approximately twice as much energy as the oxidation of carbohydrate and protein. Thus, it is not unexpected that the shrew, whose high metabolic rate and prolonged acti• vity places energy at a premium, relies heavily on this substrate to fulfill its energetic requirements. 32
^CHAPTER IV Mitochondrial Respiration of Shrew Cardiac Mitochondria 32A
INTRODUCTION
The work output of the left ventricle is defined as follows:
W = LV S-.Y,a(PLV " V (1)
where wLV = work output of the left ventricle S.V. = stroke volume PLV ~ left ventricular ejection pressure P^ = left atrial pressure
Right ventricular work is similarily defined however, due to differences in systolic pressure between the systemic and pulmonary circulation, the left ventricle in humans performs 86% of cardiac work (Guyton, 1971). Thus the work output of the heart per unit time would equal the sum of left and right ventricular work output times the heart rate.
Despite considerable species differences in arterial pressure (the long neck of a giraffe necessitates high arterial pressure in that animal to en• sure adequate blood supply to the brain) there is no systematic variation in blood pressure with body size (Altman and Dittmer, 1974). The tendency for blood pressure to increase in smaller mammals due to a decrease in capillary diameter is countered by the smaller distance through which the blood must circulate.
The allometric relationship for heart weight is as follows: 0 99 = 0.0058 \ (2) where = body mass in kilograms
= mass of the heart in kilograms
Thus heart weight equals approximately 0.6% of body mass for all mammalian species. There is some evidence (Malzahn, 1974; Bartels et_ al., 1979) that this value may be closer to 1% for some species of shrews. However, Bartels et al.-(1979) report a stroke volume of 1.1 mL/kg body mass for the Etruscan shrew. This value is similar to that of larger mammals and it appears that 33
stroke volume, like heart weight, is a constant proportion of body mass.
As stroke volume and arterial blood pressure do not vary in any systematic fashion with body size, the pressure volume work output of the heart, ex• pressed per gram of heart tissue per beat, should be constant for all mam• malian species (see equation 1). The resting rate of cardiac work pro• duction should then be proportional to resting heart rate.
Heart rate is inversely related to body size in the following manner:
-0 25 H.R. = 241 (3) where H.R. = heart rate in min
= body mass in kilograms
As body mass decreases, heart rate increases and thus cardiac work per gram per unit time must correspondingly increase.
The mechanical work done by the heart is only a fraction of cardiac energy expenditure; the rest of the energy being lost as heat. Resting cardiac efficiency is generally considered to be about 20% in mammalian hearts (Kellie and Neil, 1961), but may vary systematically with body size for the following reason. The tension in the wall of the heart (T) is re• lated to the internal pressure (P.) by Laplace's law:
T = P x R (4) where R is the radius of the heart assumed to be of a cylindrical form.
Thus, as blood pressure is approximately the same in all mammalian species, the tension in the walls of the heart will be greater the larger the radius (or size of the heart). In order for the heart to contract the cardiac muscle must develop and overcome this tension. The cardiac musculature in smaller hearts will contract at lower tensions than in larger hearts, and cardiac efficiency will thus be higher in smaller mammals. 34
This concept is demonstrated by Loiselle and Gibbs (1979) who report that, although heart rate decreases fivefold from rat to man, cardiac oxygen consumption per unit weight decreases by a factor of only two. This dif• ferences is partially attributed to the threefold increase in the energy cost per beat of cardiac contractions (from 6.5 mj/g in the rat to 20 mJ/g in man) which may be a consequence of Laplace's law.
Although the energy cost of cardiac contractions in smaller animals is reduced, does the increased rate of work output by cardiac tissue of small organisms necessitate adaptation at the cellular, physiological or biochemical level? It has been shown in the previous chapter that the abundance of mitochondria in the cardiac tissue of Sorex vagrans, although high, is within the usual range for mammalian hearts. Weibel . (1979) re• ports a cardiac mitochondrial volume of 55% for the Etruscan shrew, Suncus etruscus. This type of adaptation is limited as the proliferation of mito• chondria and associated endogenous substrate stores (lipid or glycogen) would occupy space at the expense of the tissue's "working parts", the actin and myosin complexes.
Pande and Blancher (1971) isolated mitochondria from red and white muscle of rabbit. Using palmitoylcarnitine or acetylearnitine as sub• strates, respiration rates were 80% higher in mitochondria isolated from red muscle. Also the activities of carnitine acetyltransferase and carni• tine palmitoyltransferase (expressed per milligram mitochondrial protein) were significnatly higher in red muscle mitochondria. These red muscle mitochondria thus possess a greater capacity for fatty acid oxidation than white muscle mitochondria. This would be of adaptive advantage to a muscle which functions primarily for sustained aerobic activity. 35
Palmer e_t al. (1977) have developed a technique to seperate subsarco•
lemmal (peripheral) from interfibrillar mitochondria in rat cardiac tissue.
These two populations of mitochondria show different physiological and bio• chemical properties. Interf ibrillar mitochondria oxidize glutamate, ct-keto- glut&rate and pyruvate at a faster rate than peripheral mitochondria. Also the activities of succinate dehydrogenase and citrate synthase are higher
in interfibrillar mitochondria. The authors imply that these two populations of mitochondria may fulfill different metabolic roles but do not speculate as to what these roles might be. The hypothesis of Muller (1976) that inter- fibrillar mitochondria provide the ATP necessary for skeletal muscle con• traction while peripheral mitochondria supply energy necessary for transport processes may also be applicable to cardiac tissue. Thus it is evident that mitochondria isolated from different tissues and indeed mitochondria iso•
lated from different components of the same tissue can by physiologically and biochemically adapted to function under a variety of metabolic regimes.
As the work output per gram of cardiac tissue increases with decreasing body size, it was decided to examine several physiological and biochemical properties of mitochondria isolated from shrew and rat cardiac tissue.
Specifically three questions are asked:
(1) Do the mitochondria of shrew and rat hearts differ in their absolute respiration rates when incubated with various metabolic substrates?
(2) Do the mitochondria differ in substrate preferences in such a way that might indicate an emphasis on carbohydrate or lipid as a primary energy source?
(3) Are there any differences in the enzymatic pro• files of shrew and rat heart mitochondria? 36
RESULTS AND DISCUSSION
Table 1 summarizes the respiration rates of mitochondria isolated from shrew and rat mitochondria. Using malate and pyruvate as substrates, oxi• dation rates appear slightly higher in shrew mitochondria. Rates obtained using malate.and palmitoyl carnitine as substrates show no significant dif• ference between the two organisms (at 200 yM ADP). Under both substrate conditions doubling the ADP concentration (to 400 yM) increases the state 3 respiratory rates to a greater extent in shrew mitochondria than rat mito• chondria. A comparison of mitochondrial oxidation rates at ADP concen• trations less than 200 yM (range tested 20-200 yM) did not reveal any dif• ferences between the shrew and rat. Respiration rates using glutamate, succinate, acetylcarnitine and a-ketoglutamate as substrates were also determined. Although ADP/0 ratios and respiratory control ratios using these substrates are considerably lower than those given in Table 1, the rates obtained for each substrate are similar for both shrew and rat cardiac mitochondria.
Table 2 gives the activity of various enzymes in mitochondria prepared from shrew and rat hearts. Lactate dehydrogenase is included as an indi• cator of cytoplasmic contamination. LDH activity is low and, as some LDH might always be present bound to the outer mitochondrial membrane (Suarez, pers. comm.), it is evident that cytoplasmic contamination is slight.
Citrate synthase, and B-hydroxy butyrylCoA dehydrogenase levels are approxi• mately 60-70% higher in shrew mitochondria. MDH and GOT levels are 100% higher in rat mitochondria while GDH and fumarase levels are similar in both species.
Malate dehydrogenase and glutamate-oxaloacetate transaminase have both 37
TABLE I
Respiration rates of mitochondria from heart tissue of the shrew and rat.
Values are means +_ the standard error (S.E.), expressed as nanomoles of
consumed/min/mg mitochondrial protein. Assay conducted at 25°C, pH 7.4.
Detailed assay conditions are given in Materials and Methods. Shrew re• sults are from three separate mitochondrial preparations, rat values are means of four preparations (*N = 2). Respiratory control ratios range between 2.68-3.95 while ADP/O ratios are between 2.02 and 2.73. Protein concentration in the incubation vessel was approximately 0.2 mg. 37A
Substrate Oxidation Rate
Shrew Rat
5 mM Malate 5 mM Pyruvate 159 + 3 135 + 4 200 m ADP
5 mM Malate 5 mM Pyruvate 189 + 8 142 + 1* 400 yM ADP
5 mM Malate 30 yM Palmitoyl Carnitine 183 + 14 171 + 5 200 yM ADP
5 mM Malate 30 yM Palmitoyl Carnitine 208 + 17 152 + 9* 400 yM ADP 38
TABLE II
Enzyme activities in mitochondria prepared from shrew and rat hearts.
Mitochondrial preparations were frozen and thawed prior to homogenation to facilitate the release of membrane-associated enzymes. Values are
+ standard error, expressed as u moles of substrate converted/min/mg mito• chondrial protein at 37°C. Detailed assay conditions are given in
Materials and Methods. Lactate dehydrogenase was assayed as an indicator of cytoplasmic contamination. Sample size was four for both organisms.
(*N = 3). 38A
Enzyme Activity
Shrew Rat
Citrate synthase 2.63 + 0.07 1.49 + 0.01
Fumarase 2.58 + 0.46 2.14 + 0.12
Malate dehydrogenase 11.2 + 0.5 21.4 + 0.1
Glutamate-oxaloacetate 2.45 + 0.26 4.04 + 0.09 transaminase
Glutamate 0.08 + 0. 0.06 + 0.01* dehydrogenase
6-hydroxy butyrylCoA 4.33 + 0.23 2.74 + 0.07 dehydrogenase
Lactate dehydrogenase 0.13 + 0.04 0.08 + 0.03 39
a mitochondrial and cytoplasmic form. One of the principle functions of both enzymes is the transfer of cytoplasmic NADH, produced by aerobic gly• colysis, into the mitochondria via the malate-aspartate shuttle (Hochachka,
1980). If shrew heart does not rely on aerobic glycolysis to fulfill its energetic.requirements, but rather utilizes primarily blood-born fatty acids and endogenous lipid, all reducing equivalents would be produced and oxi• dized in the mitochondria via the fatty acid oxidation system. Neely and
Morgan (1974) report that isolated perfused rat hearts utilize fatty acids in preference to both glucose and endogenous lipid stores, and lipid stores in preference to blood born glucose. However up to 20% of the aerobic energy requirements in rat heart can be provided by glucose oxidation. A decreased requirement for the malate-aspartate shuttle, due to a reduction in the de- pendancy of shrew heart on glucose oxidation, could account for the de• creased levels of mitochondrial MDH and GOT observed in shrew cardiac tissue.
Thus it is evident that the absolute rates of cardiac mitochondrial respiration are similar in both the rat and the shrew, although shrew mito• chondria may utilize pyruvate at a slightly higher rate. In addition, cardiac mitochondria isolated from the shrew do not show any distinct substrate pre• ferences which might indicate an adaptation of shrew mitochondria towards the increased utilization of a particular metabolic pathway (i.e., fatty acid oxidation). Holian et al. (1977) report that mitochondrial respiration in dog and pigeon heart muscle is tightly controlled by extramitochondrial f_ATP3 / [ADP] XP^l. Although the adenylate translocase system is generally saturated at physiological ADP and ATP concentrations, competition between
ADP and ATP for binding sites can effect the rate of ..mitochondrial respira• tion. In the present study, ATP was not included in the incubation medium and thus it was expected that both the shrew and rat adenylate translocase systems would be saturated at 200 uM ADP (approximately the physiological 40
extramitochondrial concentration). It is difficult to interpret the increase in respiration rate at 400 uM ADP as the translocase activity may be dis• rupted during the isolation of mitochondria in such a way that it becomes rate limiting (Holian et al., 1977). It would be interesting to determine whether any intrinsic differences exist between the adenylate translocase systems of the shrew and rat which might lead to an inherently higher rate of respiration in the shrew mitochondrion. This type of study would re• quire detailed.competition experiments to determine the effect of varying
[ATP] / [ADP][Pi] ratios on mitochondrial respiration.
Smith (1956) reports that as body mass decreases the number of mito• chondria per gram of liver increases such that the total mitochondrial mass scales in proportion to basal metabolism. In addition oxygen utilization by liver homogenate is shown to depend more directly on abundance of mito• chondria per gram than on the unit mass of mitochondrion. The results of this current study do not show a startling difference in mitochondrial abundance.between rat and shrew cardiac tissue (Chapter II) nor a signi• ficant difference in the mitochondrial respiration rate expressed per mg of mitochondrial protein. This does not preclude the possibility that the cardiac mitochondria of shrews and rats have evolved to exploit different metabolic frameworks. Indeed if the amount of protein (enzyme) per cardiac mitochondria is greater in shrews, then identical respiration rates (ex• pressed per mg mitochondrial protein) implies that the respiration rate per unit mitochondrion is greater in shrews than rats.
The enzymatic profiles obtained for shrew and rat mitochondria (Table 2) indicates that inherent differences may in fact exist. Shrew mitochondria exhibit elevated levels of the key Krebs cycle enzyme, citrate synthase, as well as increased levels of 8-OH butyrylCoA dehydrogenase, an enzyme of the 41
fatty acid oxidation spiral. The abundance of storage lipid in cardiac tissue has been previously noted (Figure 1) and it is probable that shrew cardiac muscle relies heavily on fatty acid oxidation to supply its ener• getic requirements under all physiological conditions. 42
CHAPTER V
Enzyme Profiles in Various Shrew Tissues 42A
INTRODUCTION
The preceding chapters have outlined a number of morphological and biochemical adaptations of shrews which appear to be related to their in• trinsically high basal metabolic rate. Ultrastructural studies indicate an abundance of mitochondria and storage lipid in heart, liver, diaphragm and gastrocnemius muscle. Although no large differences could be observed in the rates of oxidation by cardiac mitochondria isolated from rats and shrews, the enzymatic profile of shrew mitochondria appears to be more closely geared to fatty acid oxidation than rat mitochondria.
Kuntel and Campbell (1952) and Simon and Robin (1971) have reported an inverse relationship between cytochrome oxidase activity in various vertebrate tissues and body size. The former authors show that the acti• vity of this enzyme in liver and skeletal muscle increases as body size decreases. No correlation with body size was demonstrable for brain and kidney cytochrome oxidase. The latter authors demonstrate an exponential relationship between myocardial cytochrome oxidase activity and mass specific oxygen consumption. Fried and Tipton (1953) observed an inverse relationship with body size of the activities of succinate dehydrogenase and malate dehydrogenase in various tissues of cow, rat and mouse. The activities of several enzymes of amino acid metabolism (phenylalanine- pyruvate, phenylalanine-a-ketoglutarate, tyrosine-pyruvate and tyrosine- a-ketoglutarate transaminases) in liver scale with body size in approxi• mately the same manner as basal metabolism (Lin et^ al_. , 1959) .
In the present study maximal enzyme activities of key respiratory en• zymes in the heart, liver and skeletal muscle of the shrew were determined and compared to corresponding tissues in the laboratory rat. The purpose 43
of this investigation was twofold; (i) to determine which metabolic path• ways, if any, are enhanced in shrew tissues and (ii) to determine if there is a general increase in respiratory enzyme titers in the shrew, presumably related to its very small size and high basal metabolic rate.
The literature contains numerous examples of maximal enzyme levels in various mammalian tissues, including the rat (Scrutton and Utter, 1968; Alp et al., 1976; Crabtree and Newsholme, 1972a; Crabtree and Newsholme, 1972b;
Zammit e_t. al_., 1978). It is however, difficult to compare the results of one investigation with another as isolation techniques and assay methods are conducted using different conditions of pH, buffer composition, tem• perature or substrate levels. In addition, activities are often ex• pressed in a gammut of units, which are not readily interconvertible. To alleviate the difficulty of obtaining comparable literature data, enzyme levels in the rat were determined concurrently with the shrew assays.
RESULTS AND DISCUSSION
Heart
Table 3 summarizes the enzyme activities in both shrew and rat heart, liver and skeletal muscle. Shrew heart contains only 20% of the total phosphorylase activity recorded for rat heart. The total phosphorylase assay includes AMP to activate the enzyme and is a more useful measure of the potential for a tissue to metabolize glycogen than an assay con• ducted without the addition of AMP. This reduced phosphorylase activity correlates well with scarcity of glycogen found in the tissue sections of shrew heart. Hexokinase levels are also depressed with respect to rat heart, indicating a decreased reliance on blood-born glucose as an energy 44
TABLE III
Enzyme activities in the heart, skeletal muscle and liver of the shrew
and rat. Skeletal muscle activities were determined from homogenates of
'hindlimb musculature of the shrew and gastrocnemius muscle of the rat.
Values are means _+ standard error, expressed as p moles of substrate con-
verted/min/g wet weight at 37°C. Detailed assay conditions are given in
Materials and Methods. Sample size is four (*N = 3). N.D. = not de•
tectable. Enzyme Heart Skeletal Muscle Liver
Shrew Rat Shrew Rat Shrew Rat
Glycogen 3.4+0.4* 12.8+2.0 8.6 + 3.3* 54 + 11* 1.87 + 0.31* 4.15 + 1.89 phosphorylase
Total 7.4+0.9* 37.0+5.7 11.7 + 6.4* 91 + 13* 3.93 + 0.27* 5.63 + 2.72 phosphorylase
Hexokinase 1.7+0.5* 3.1 +0.5 0.41 + .09* 0.24 + .03* N.D. at N.D. at 1 mM glucose 1 mM glucos
Pyruvate 333 + 8 245 + 8 179 + 29 842 + 48 65 + 5 111 +4 kinase
Lactate 904 + 53* 951 + 43 165 + 23 1206 + 70 411 + 25 707 + 5 dehydrogenase
Citrate 334 + 18 128 + 4 74 +6 19.6 + 2.7 25.7 + 0.5 5.3 + 0.6 synthase
Fumarase 348 + 12 147 + 15 104+9 33+4 439 + 14 118 + 6
Malate 2577 2816 + 84* 1002 + 99* 907 1162 + 96* 837 + 45 dehydrogenase (n=2) (n=2) TABLE III (CONTINUED)
Enzyme Heart Skeletal Muscle Liver
Shrew Rat Shrew Rat Shrew Rat
Glutamate- 541 + 20 356 + 7 177 + 11 118 430 + 41 104 + 4 oxaloacetate (n=2) transaminase
Glutamate- 11.5 + 1.3 44 + 4.4 ,0 + 3.2* 41 + 13* 129 + 7 120 + 10 pyruvate transaminase 3-hyroxyl 428 + 27 223 +10 87+6 17.9 + 4.5 185 + 7 98 + 12 butyrylCoA dehydro• genase
Carnitine 0.57 + .09 0.57 + .06 0.17 + .05 0.01 + .01 palmitoyl trans• ferase 45
substrate. Levels of PK are somewhat greater in shrew heart and no signifi•
cant difference is observed in LDH levels. Recent evidencew(Liu and Spitzer,
1978) suggests that lactate may be preferentially oxidized by the heart whenever concentrations rise above normal levels. Thus high LDH levels in shrew heart are perhaps as involved in the oxidation of lactate produced by the peripheral organs as in lactate production in_ situ by anaerobic gly• colysis. On the other hand, the relatively high level of pyruvate kinase in the shrew heart implies a high capacity for the glycolytic metabolism of triose substrate. As shrew metabolism invests heavily in fat, the glycerol released from triglyceride hydrolysis might serve as the triose substrate requiring the maintenance of high titres of enzymes such as PK in the lower half of the glycolytic pathway.
Activities of citrate synthase and fumarase are two to three times higher in.shrew heart. The activity of citrate synthase in shrew heart is higher than all mammalian and avian heart citrate synthase values reported by Alp e^t al_. (1976) when corrected for the different assay temperatures by assuming a Q^^ of 1.2 this enzyme, as reported by the above authors. In• deed the citrate synthase level in shrew heart approaches values recorded for insect flight muscle. As MDH levels in shrew cardiac mitochondria are less than rat mitochondrial levels (Table 2), cytoplasmic activities of MDH must be greater in the shrew to account for the equal cellular levels ob• served in Table 3.
The activity of carnitine palmitoyl transferase is the same in both rat and shrew hearts, however levels of B-OH butyrylCoA dehydrogenase, an enzyme of the fatty acid oxidation spiral, are almost twofold higher in shrew hearts. Although cardiac mitochondrial content is perhaps slightly higher in shrew hearts (Chapter II), the increased levels of citrate syn- 46
thase, fumarase, and B-OH butyrylCoA dehydrogenase observed in shrew cardiac tissue may also be due to differences in the actual concentration of oxida• tive enzymes in the mitochondria of shrews and rats.
As is the case with MDH, although mitochondrial levels of GOT are less
in the shrew than rat (Table 2), overall tissue levels are higher in shrews.
In contrast, the activity of glutamate-pyruvate transaminase is less in
shrew cardiac tissue. GPT is involved in the production of alanine from pyruvate during the metabolism of branched chain amino acids by cardiac and
skeletal muscle (Goldberg and Chang, 1978). As the pyruvate in this pathway
is ultimately derived from glucose, perhaps the reduced level of aerobic glycolysis in shrew heart does not provide adequate substrate for this pathway to operate extensively.
Liver
The enzyme profiles of rat and shrew liver follow the general pattern
of increased activities of aerobic enzymes and decreased levels of anaerobic
enzymes observed in shrew cardiac tissue. Citrate synthase, fumarase and
B-OH butyrylCoA dehydrogenase activities are again higher in shrew tissue, while levels of PK and LDH as well as phosphorylase are depressed. Hexo- kinase is not normally detectable in liver, although glucokinase activity
(at non-saturating glucose concentrations) was greater in the shrew liver.
Large lipid stores were observed in the electron micrographs of shrew hepa-
tocytes and it appears that liver as well as heart has an enhanced ability
to metabolize this substrate.
It is of interest to note the different proportions of Krebs cycle
enzymes present in liver as compared to cardiac and skeletal muscle. Both muscle tissues have quite similar proportions of citrate synthase: fumarase: 47
B-OH butyrylCoA dehydrogenase (approximately 1:1.5:1). Both shrew and rat liver levels of citrate synthase are reduced to about 6% of fumarase levels.
Ultrastructural studies also show a striking difference in the morphology of hepatic and muscle mitochondria. The inner mitochondrial membrane of the former is not as highly fenestrated, and presumably affords less surface area for membrane-associated functions.
Skeletal Muscle
The results summarized in this section could perhaps be construed as misleading because the entire hindmusculature of the shrew was assayed for enzyme activity and compared to gastrocnemius muscle of rats. Unfortunately, shrew gastrocnemius did not afford an adequate homogenate volume to assay all the required enzymes. It was necessary to use solely gastrocnemius muscle in the rat as these enzyme profiles were subsequently to be compared to gastrocnemius profiles from larger mammals.
Skeletal muscle displays the most dramatic shift from a highly glyco• lytic tissue in the rat to a seemingly lipid-based aerobic metabolism in the shrew. Phosphorylase, pyruvate kinase, and lactate dehydrogenase acti• vities in shrew skeletal muscle are 10-20% of values recorded for rat gastro• cnemius, while citrate synthase, fumarase, and B-OH butyryCoA dehydrogenase levels are increased three to four fold in the shrew. GOT and GPT levels follow the same pattern as observed for cardiac muscle. Carnitine palmitoyl transferase, the enzyme responsible for the transfer of long chain fatty acids from the cytosol into the mitochondria, is also present at higher levels in shrew skeletal muscle.
The enzyme activities of shrew skeletal muscle recorded in the present study correspond quite well to values reported by Beatty and Bocek (1970) 48
for various red muscles of several mammalian species. The data reported by the authors are however a synthesis from a variety of studies in which activities are expressed by a plethora of units and the assay temperatures are not stated, and thus a quantitative comparison is impossible.
Those enzymes whose activities are increased dramatically in shrew skeletal musculature are all located in the mitochondria (citrate synthase, fumarase, B-OH butyrylCoA dehydrogenase). It has been seen (Chapter III) that shrew gastrocnemius contains abundant mitochondria, particularly in the subsarcolemmal region. The enzyme data suggests that the same prolifer• ation of mitochondria exists throughout the hindlimb musculature of the shrew.
Scaling of Gastrocnemius Enzyme Activities
As such a dramatic difference was observed in the enzyme profiles of shrew and rat skeletal muscle, it was decided to extend this study over a wider range of mammalian species to determine if any systematic variation in the enzyme profiles of gastrocnemius muscle with respect to body size is demonstrable.
Table 4 summarizes the enzyme profiles of gastrocnemius muscle from seven mammalian species ranging from the 4 gram shrew, Sorex vagrans, to the 370 kilogram cow. It is evident that, as animal size decreases, the activities of the glycolytic enzymes (PK, LDH) and phosphorylase decrease while the indicator enzymes for the Krebs cycle (citrate synthase) and fatty acid oxidation (B-OH butyrylCoA dehydrogenase) increase in activity.
Malate dehydrogenase levels also appear negatively correlated with body weight, although the relationship is less dramatic than the other oxidative enzymes, probably because this enzyme may have dual (aerobic and anaerobic) functions. 49
TABLE IV
Enzyme activities in the gastrocnemius muscle of animals of varying size.
Due to the small size of the shrew it was necessary to use the entire hindlimb musculature to assay enzyme activity. In all other cases, only the gastrocnemius, or medial portion thereof, was used for the enzyme assays. Activity is expressed as umoles of substrate converted/min/gram wet weight at 37°C. Detailed assay conditions are given in Materials and
Methods. Each valuerepresents the mean of duplicate assays. Animal Weight Enzyme Activity
(g) Glycogen Total Pyruvate Lactate Citrate Malate 3-OH ButyrylCoA phos- phos• kinase dehydro• synthase dehydro• dehydrogenase phorylase phorylase genase genase
Sorex vagrans 4.4 12.4 20.4 129 138 61 979 75 (shrew) 5.4 11.3 21.3 163 178 69 1 185 86
Peromyscus 13.3 31 49 320 682 34 745 62 maniculatus 14.7 11.0 66 415 690 56 935 77 (Deer mouse)
Microtus 54.1 68 71 709 1 022 27 684 7.1 townsendii 58.3 64 68 655 991 25 672 7.2 (vole)
Rat 344 78 112 862 1 144 27 1 085 30 384 55 94 717 1 075 17.3 728 18
Guinea Pig 510 11.1 53 432 862 23 528 18 710 19.3 53 442 946 28 690 21
Rabbit 1 900 58 75 958 2 209 7.0 306 4.6
Cow 3.7 x 10' 7.0 86 666 2 146 7.2 485 6.6 50
As stated previously, gastrocnemius is a mixed muscle, composed of both red and white fibers. Considerable species variation in the fiber composition of this muscle exists, often related to the energetic require• ments of the particular species. For example, Schmidt-Nielsen and Pennycuik
(1961) report cat gastrocnemius to be composed of 100% white fibers while the same muscle in dog is made up of 100% red fibers. Canines can function for long periods of time at high aerobic work rates while cats generally capture prey by a rapid burst of anaerobic muscular work. In the present study, the enzymatic profile of the rabbit, also a burst worker, is not dramatically different from that of the much larger cow. As well as such individual variation there appears to be, at least in gastrocnemius muscle, a systematic increase in the enzyme activities characteristic of red muscle fibers as body size decreases.
Figure 9 is a log/log graph of citrate synthase activities showed in
Table 4 plotted against body mass. Using linear regression, the relation• ship between the enzyme activities and body mass can be expressed as follows:
Activity of citrate synthase = 72.4
where equals body mass in grams.
A similar plot of lactate dehydrogenase activity against body mass (Figure
10) is expressed mathematically as follows:
Activity of LDH = 269 l^0'21
As outlined in the Introduction, the scaling of metabolic turnover rate
is often expressed as:
25 = 0.696 Mb-°-
-1 -1 where VQ /M^ = oxygen consumption in liters C^ h kg 2 = body mass in kilograms
Conversion of this equation to units of grams and minutes would change the 51
FIGURE 9
Activity of citrate synthase in the gastrocnemius muscle of several mam• mals as a function of body size. Data from Table 4. Equation for the solid line, determined by linear regression, is A = 72.4 M^-^'2^ where
A = activity of citrate synthase and = body mass in grams.
The regression line of log (V02max/Mb) versus (Taylor et_ al_., in pres is added as a dashed line for comparison. ENZYME ACTIVITY ( jimoles substrate/min/g wet weight)
v O-mcs/Mb (ml 0- kg-'s-l)
vie 52
FIGURE 10
Activity of lactate dehydrogenase in the gastrocnemius muscle of several mammals as a function of body size. Data from Table 4. Equation for the 0 21 solid line, determined by linear regression, is B = 269 " where B = activity of lactate dehydrogenase and = body mass in grams. ENZYME ACTIVITY (ymoles substrate / min / g wet weight) 53
value of the coefficient but the scaling exponent (-0.25) would remain con• stant. Thus the aerobic enzyme citrate synthase in gastrocnemius muscle varies with respect to body mass with approximately the same exponent as metabolic turnover rate. Lactate dehydrogenase, on the other hand, scales with equal magnitude but in a positive fashion with body mass.
The variation in skeletal muscle structure and function with respect to body size has not been extensively studied. Gauthier and Padykula (1966), in a study of the diaphragm muscle of 36 mammalian species, demonstrate an increase in the proportion of smaller red fibers and in mitochondrial con• tent as body size decreases. Davies and Gunn (1972), in a histochemical study, report that the diaphragm of smaller animals is composed primarily of fast-twitch fibers of limited anaerobic capacity, while the diaphragm of larger animals has a majority of slow twitch fibers, capable of both aerobic and anaerobic metabolism. However, no systematic variation in succinate dehydrogenase content, indicative of changing mitochondrial den• sity, was observed. The same authors, in a similar histochemical study of semitendinosus muscle (Davies and Gunn, 1971), also observe an increase in the proportion of fast twitch fibers as body size decreases, although the effect is not as pronounced as in the diaphragm.
Some interesting insights have been shed on this problem in a recent study of mitochondrial abundance in the skeletal muscle of a series of
African mammals ranging in body mass from 0.4-251 kilograms (Mathieu et al., in press). These authors studied the scaling of mitochondrial abundance in four skeletal muscles. The volume density of mitochondria (i.e., the fraction of a given tissue volume occupied by mitochondria) scaled as -0.231 -0.163 -0.139 , -0.055 . . M, , M, , M, and M, m semitendinosus, longissimus dorsi, vastus medialis, and diaphragm respectively. The authors hypothesis 54
that, in terrestrial mammals, it is likely that the total volume of muscle mitochondria should scale in proportion to maximal oxygen consumption, as skeletal muscle will consume up to 96% of the oxygen taken up by the lungs under conditions of maximal work loads (Weibel, 1979). Concurrent studies by Taylor ejt al_. (in press) shows that mass specific maximal oxygen con• sumption ^Q^max/U^) in this group of African mammals scales as M^"^'2^.
The fraction of total muscle volume occupied by mitochondria was then calculated, thus accounting for the small scaling factors of individual muscle mass with body size. It was found that these absolute mitochondrial volumes vary with body size with approximately the same exponent as maximal oxygen consumption (Vg^max).
In the present study the activity of the mitochondrial enzyme citrate synthase also scales with approximately the same exponent as VQ max/M^, while lactate dehydrogenase activity scales in an opposite fashion to oxidative enzyme activities and maximal oxygen consumption. Table V sum• marizes confidence limits for the regression lines of citrate synthase,
B-OH butyrylCoA dehydrogenase and LDH activities versus body mass. Al• though the difference in the scaling exponents of citrate synthase (-0.20) and basal metabolic rate (-0.25) is not significant at the 95% confidence level, oxidative enzyme activities do scale with exponents closer to
Vo9max/M, than VQ /M, . A larger sample size would give a better indication of the statistical significance of this observation.
However the scaling exponents of citrate synthase and B-OH butyrylCoA dehydrogenase suggest that gastrocnemius mitochondrial volume also varies with respect to body size in approximately the same fashion as observed by
Mathieu et_ al_. (in press) . In order to gain further insights into the allometric relationships for skeletal enzyme activities and mitochondrial 55
TABLE V
Statistical analysis of linear regressions of enzyme activity versus body mass. Equations are of the form x = AM^ where x equals enzyme activity,
= body mass in grams, A = coefficient and B = regression exponent. Enzyme Coefficient (A) Exponent (B) Correlation Standard Error 95% Confidence Number Coefficient (r) of Regression Limits for of Exponent Regression Exponent Animals
Citrate 72.4 -0.20 0.88 0.034 Synthase 0.077 12
Lactate 269 0.21 0.78 0.053 0.118 12 Dehydrogenase
3-OH ButrylCoA 70.8 -0.23 0.69 0.077 0.173 12 Dehydrogenase 56 content, it would be useful to extend these studies to an examination of total skeletal musculature over a wide size range of mammalian species. 57
CHAPTER VI
Discussion 57A
It is evident that the metabolism of the shrew is adapted at a number of levels to accomodate the extremely high metabolic rate of the organism.
As this intrinsically high basal metabolic rate is a result of the ex• tremely small size of this mammal, it is impossible to discuss the bio• chemistry of Sorex vagrans without some treatment of the metabolic con• sequences of scaling in animals. Historically the scaling of oxygen con• sumption in homeotherms has been attributed to the increase in surface area/volume ratio as animal size decreases (Rubner's surface rule). This would necessitate higher basal metabolic rates in smaller mammals to com• pensate for the increased rate of heat loss. However, as stated in the
Introduction, the basal metabolic rate of poikilotfrerms- and unicellular organisms scales with the same exponent as birds and mammals and this ex• ponent (0.75) is larger than would be expected from a simple consideration of heat loss due to increased surface area (0.67).
A number of investigations have shown variations in physiological and morphological parameters which enhance substrate and oxygen delivery to and from the tissues of smaller animals. Again these studies have been reviewed in the Introduction to this thesis.' These investigations indicate that not only heat loss but other physiological processes such as oxygen uptake, oxygen and substrate transport and unloading at the tissue level are surface-related and modified in smaller animals. Thus variations in size will affect these processes. Despite the broad interest in the scaling of basal metabolism, the consistent regression of basal metabolic rate with, body mass in most animals and even some tree species to give a linear relationship with a slope of 0.75 remains largely unexplained
(Schmidt-Nielsen, 1979).
A second historical approach to study of the scaling of metabolic rate 58
has been the examination of the metabolism of tissue slices iii vitro.
Krebs (1950) examined the scaling of 0^ metabolism in brain, kidney, spleen and lung tissue of nine mammals ranging in size from a mouse to a horse.
Although a general trend towards increased 0^ consumption with decreasing animal size was noticed for all tissues (particularly liver), in no .case did tissue metabolism scale to the same extent as basal metabolic rate.
The author.suggests that skeletal muscle, which was not examined, might per• haps scale in parallel with basal metabolic rate. Similar results were obtained by Bertalanffy and Piroznski (1953) using brain, kidney, liver, diaphragm, thymus and lung slices from rats of varying size. Only the
scaling of oxidation rates of diaphragm muscle showed a close correlation with basal metabolic rate. However, a subsequent study on the scaling of mass specific oxygen consumption in skeletal muscle (Bertalanffy and
Estwick, 1953) showed this tissue to scale to the -0.07 power of body mass
in rats ranging in weight from 10 to 300 grams. In contrast the scaling
factor for metabolic turnover rate is -0.25. The authors conclude that, contrary to the speculation of Krebs, the scaling of skeletal muscle meta• bolism does not correlate with metabolic turnover rate any more closely than the scaling of oxygen comsumption in other tissues.
Martin and Fuhrman (1955) compared the summed tissue respiration and the basal metabolic rate of a dog and mouse. By multiplying the tissue respiration rate by the organ mass and summing for all the tissues in the body, the authors were able to account for 66% of the basal metabolic rate
of the mouse and 88% of the BMR of the dog. A similar calculation, con•
ducted by Bertalanffy and Pirozynski (1953), accounts for 66% of the BMR
of a mature rat but only 35% of a young 10 gram rat. The former authors
conclude that, as the ratio of mass of internal organs (liver, kidney and 59 brain) to total body mass increases in smaller animals, these organs con• tribute a higher proportion of basal metabolic rate in smaller animals.
According to the tabulated values of tissue respiration given by the authors, these organs account for 20% of the BMR in mice but only 17% in the dog. Similar calculations show that skeletal muscle accounts for only
26% of the BMR of the mouse and 61% of the BMR of the dog.
Skeletal muscle mass constitutes approximately 45% of total body mass for all mammalian species, irregardless of size (Munro, 1969). Elsewhere in this thesis, it has been stated that:
(i) fiber diameter of certain skeletal muscles decrease with decreasing body size (Gauthier and Padykula, 1966; present study).
(ii) enzymes of oxidative metabolism increase in skeletal muscle with decreasing body size (Kuntel and Campbell, 1952; present study).
(iii) mitochondrial volume in skeletal muscle varies inversely with body size (Mathieu et_ al., in press).
In addition, Munro and Grey (1969) report that muscle cell mass (wet wt. of muscle/mg.DNA) increases from small to large animals indicating that,
on the average, muscle cells are smaller in smaller organisms.
All of these studies indicate that oxidative metabolism is enhanced in
the skeletal muscle of smaller mammals. Thus, if skeletal muscle is a con•
stant proportion of body size, it appears counter-intuitive that the con•
tribution of skeletal muscle-metabolism to basal metabolic rate should de•
crease in animals of decreasing size. Thus it is probable that the pro• portion of basal metabolic rate unaccounted for by the tissue slice experi• ments of Martin and Fuhrman (1955) can be assigned to skeletal muscle and
that the actual contribution of skeletal muscle metabolism to basal meta• bolic rate rises as body size decreases. 60
It is notoriously difficult to prepare a proper tissue slice with skeletal muscle and it is likely that oxygen consumption rates derived from slices of skeletal muscle to not reflect in vivo rates of tissue oxygen consumption as accurately as do slices from other tissues. In addition, skeletal muscle displays a great capacity for morphological and physio• logical adaptation to enhance substrate and oxygen delivery to the tissues
(degree of capillarization, variations in muscle cell size and diameter, complexity of the T-system of the sarcoplasmic recticulum). As suggested by Krebs (1950) the resting activity of skeletal muscle iii vivo probably bears very little relationship to the metabolism of skeletal muscle slices as such "basal" activities as the maintenance of muscle tone are unaccountable in isolated tissue slice studies. It is thus evident that attempts to relate in vitro metabolism of tissue slices to the in_ vivo rate of basal.metabolism will inevidably generate equivocal results and the conclusions of these studies should be regarded skeptically.
A third approach to the study of the scaling of metabolic rate has been at the biochemical level. This aspect of metabolic scaling has not been explored to any great extent and further work in this area may add new in• sights to our understanding of the scaling phenomenon. We have seen that
\iax values ^° tend t0 increase as body size decreases (Kuntel and Campbell,
1952; Simon and Robin, 1971; Fried and Tipton, 1953; Lin et al.,.1959, present study). However, these differences in enzyme activity are generally less than the changes in mass specific metabolic rate.
Although the activities of oxidative enzymes are higher in the shrew than the rat in all tissues examined, by far the greatest differences are seen in skeletal.muscle. Furthermore, the levels of enzymes of the Krebs cycle and fatty acid oxidation in gastrocnemius muscle appear to scale with 61
body mass to the same degree as maximal oxygen consumption. Using the relationship of Taylor e_t al. (in press) for Vg^max and body mass and the allometric relationship for basal oxygen consumption given in Chapter I, one can calculate that a four gram shrew maintains the capacity to in• crease aerobic metabolism approximately seven times above basal while a
500 kilogram horse displays a scope for aerobic metabolism of about 12 times. The difference in aerobic scope is due to the fact that basal metabolism scales as the 0.75 power of body mass while VQ^max scales as the
0.79 power of body mass. Thus the absolute concentrations of oxidative enzymes in shrew skeletal muscle must be at levels where the capacity to increase metabolic flux by a factor of seven is maintained. The horse on the other hand must maintain the enzymatic capacity to increase flux rates twelve times above basal. Scaling of oxidative enzyme activities in parallel with VQ^max/M^ ensures that flux rates can be increased to these levels.
The present study indicates this relationship between oxidative enzyme activity and Vg^max for gastrocnemius muscle. The scaling of mitochondrial
abundance in parallel with Vn max (Mathieu et al., in press) suggests that u2 the relationship also holds true for semitendinosus, longissimus dorsi and vastus, medialis muscles. It would be of interest to examine the re• lationship between oxidative enzyme activities and ilQ^max in total skeletal musculature of terrestrial mammals.
In accordance with Michaelis-Menten kinetics, the increase in flux rate during the transition for resting to active metabolism is achieved by adjusting the enzymatic potential through changes in effective enzyme concentration or by increasing substrate levels. Both mechanisms apparently are utilized. In mammalian muscle increased flux through a metabolic path- 62
way is associated with rising levels of metabolic intermediates. Electrical stimulation of single white muscle fibers of rat hindlimb musculature for one minute results in a 15 fold rise in the level of glucose-6-phosphate and a seven fold increase in fructose-6-phosphate levels (Lowry, pers. comm.). Sacktor et al. (1965) measured increases in the levels of pyruvate and a-glycerophosphate during the electrical stimulation of in. situ rat muscle. Increased flux rates are primarily achieved through the activation of regulatory enzymes by metabolic effectors, resulting in an increase in the effective enzyme concentration at key control points of the metabolic path• way. Activation of glycogen phosphorylase by phosphorylation, phospho- fructokinase by FDP and ADP binding, and citrate synthase by a reversal of
ATP binding are a consequence of effector-induced changes in the Michaelis constant (k ) and/or turnover number (k ) of the enzyme. This results m cat in a much larger portion of the enzyme molecules being active at approxi• mately the same substrate concentration which existed prior to activation of the enzyme. The overall "on-off" mechanism of metabolic flux control by regulatory enzymes ensures that, at all flux rates, most enzymes are
working at substrate levels much less than their respective km values.
Thus the enzymes function on that portion of the Michaelis-Menten curve where the greatest velocity changes will occur for an incremental change in substrate level, thus affording an effective second level of metabolic control. The rise in the levels of certain metabolic intermediates is necessary to ensure that flux rates through non-regulated, equilibrium reactions of the pathway are also increased.
In one extreme case, the flight muscle of insects, substrate levels do not rise significantly when flux rate through glycolysis is increased.
In the blowfly, oxygen consumption is increased approximately 100 fold upon 63
the initiation of flight. The energy for the intensely respiring flight muscle is largely provided by the oxidation of glycogen and trehalose via the glycolytic pathway and Krebs cycle. Despite a momentary increase in substrate levels upon the initiation of insect flight, metabolic inter• mediates of glycolysis and the Krebs cycle remain at approximately the same levels before and during flight (Sacktor and Wormser-Shavit, 1966).
This result can only be due to very tight regulation of all the enzymes involved in the oxidation of these substrates. Recent evidence suggests that Ca++ may exert a significant role in the regulation of enzymes of insect flight muscle (Sacktor, 1976).
It is tempting for the biochemist to speculate that, as body size de•
creases and mass specific 02 consumption or metabolic turnover rate as de• fined by Kleiber (1975) increases, it would be of selective advantage for key regulatory enzymes to "tick over" faster in smaller animals. In parti•
cular, one might expect that k t, the number of substrate molecules con• verted to product by an enzyme molecule per unit time, would be larger in the enzymes of smaller organisms.
As enzyme activities ,are normally measured under saturating substrate conditions, these activities,V^^,will consist of two components; total enzyme concentration V_E "\ , and the turnover number of the enzyme (k ). O Celt Thus any systematic change in k values with respect to body size would cat
be reflected in ^max measurements but, without purifying the particular enzyme to absolute homogeneity and measuring the turnover number directly,
it is impossible to know if ^max differences are due to changes in enzyme concentration or k . However comparison of the results of the present Celt study with the scaling of mitochondrial abundance in skeletal muscle
(Mathieu et^ jQ., in press) indicates that skeletal oxidative enzyme acti- 64
vities.scale in parallel.with mitochondrial abundance. As these enzymes are located in the mitochondria it is probable that smaller animals have higher absolute concentrations of oxidative enzymes.
We have seen that activation of mammalian skeletal muscle is accom• panied by increases in the levels of metabolic intermediates. Thus it is also possible to speculate that resting substrate levels are higher in small animals. Osmotic considerations and the fact that metabolic regulation is best effected when substrate levels are less than k limit the possi• m bilities of this mode of metabolic rate enhancement. However no systematic study of the variation of tissue levels of metabolic intermediates and body size exists. A single study by Umminger (1975) suggests that resting blood glucose levels rise as animal size decreases.
In summary the activities of oxidative enzymes appear to increase as animal size decreases. This is particularly true for skeletal muscle, in which the activity of citrate synthase scales with approximately the same
exponent as Vo?max/M, .
As well as the observation that oxidative enzyme activities scale in parallel with maximal oxygen consumption, a second interesting aspect of the present study is the increase in anaerobic enzyme activities with in• creasing body mass. Again this observation is most pronounced in skeletal muscle (gastrocnemius) where LDH scales in an exact opposite fashion to the scaling of citrate synthase and B-OH butyrylCoA dehydrogenase levels
(Figures 9 and 10). These increases in phosphorylase, PK, and LDH activities with increasing body size suggests that burst anaerobic work is functionally more important in larger animals.
Even if a small animal did possess the same capacity for burst work as a predator larger than itself, it would still be unlikely to escape the 65
predator in a chase powered by burst anaerobic work. Thus smaller animals rely on the ability to conceal themselves from predators, rather than the strategy of the antelope, which can outdistance its pursuer in a burst of anaerobic work.
Coulson et^ al_. (1977) provide an interesting hypothesis as to why smaller mammals are forced to be less dependent on anaerobic metabolism.
Assuming that all animals have comparable abilities to store muscle glycogen,, the authors calculate that this glycogen, metabolized anaerobically, could provide the resting energy requirements of a shrew for a maximum of 13 seconds, but an elephant could be sustained for several hours. Under nor- moxic conditions, it is unlikely that any mammal would utilize anaerobic glycolysis to fulfill its basal metabolic needs, however it is obvious that muscle glycogen stores in the shrew provide an insufficient amount of energy for any significant burst anaerobic work. Taylor et_ aT_. (1970) have shown that the energetic cost of aerobic locomotion rises (on a mass specific basis) as mammalian body size decreases. As this energy cost is a direct consequence of the animal's size and form one would expect this to hold true for anaerobic locomotion as well. This further strengthens Coulson's argument as a greater amount of energy would have to be produced to per• form equal work in small versus large mammals.
A recent study by Somero and Childress (1980) examines the scaling of citrate synthase, MDH, PK and LDH activities in the white muscle and brain of fishes of varying size. In white muscle citrate synthase activity scales in parallel with basal oxidative metabolism, while PK and LDH in• crease in activity with increasing body length or mass. In brain tissue citrate synthase levels also scale in a similar fashion as oxidative meta• bolism; however no systematic variation in glycolytic enzyme activities are 66
observed. The authors point out that PK and LDH scale with an exponent which is close to the scaling exponent for the power requirements of burst work. In other words as a fish increases in length the power required for it to reach burst swimming speeds increases on a mass specific basis. The increased activities of PK and LDH provide the enzymatic machinery necessary to enhance the mass specific power output of the white muscle.
Thus, in both fish and mammalian muscle, aerobic and anaerobic enzyme activities scale in opposite fashions with respect to body size. The re• lationship of Taylor et.-al. (in press) for the scaling of maximal oxygen consumption (Vrj^max) shows that the aerobic scope for activity in small mammals will be less than larger mammals (as BMR scales to the 0.75 power of body mass while VQ^rnax scales to the 0.79 power). In fishes the dif• ference in aerobic scope between large and small animals is even more pronounced.. Maximal oxygen consumption in sockeye salmon (Oncorhynchus nerka) scales as the 0.97 power of body mass while basal metabolism scales as the 0.78 power of body mass (Brett, 1965). Given that skeletal muscle activities of glycolytic enzymes increase as both mammalian and piscine size increases, it is expected that the difference in basal to burst work scope between large and small members of the animal groups will be even more pronounced than the differences in aerobic scope. 67
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